Thiourea derivatives of mitomycin



Feb. 6, 1968 MASANAO MATSUl ETAL 3,367,945

THIOUREA DERIVATIVES OF MITOMYCIN Filed Dec. 1, 1965 6 Sheets-Sheet 1 F/GT I -INVENTORS %W. 4Z

ATTORNEYS Feb. 6, 1968 MASANAO MATSUI ETAL 3,367,945

THIOUREA DERIVATIVES OF MITOMYCIN Filed Dec. 1, 1965 e Sheets-Sheet 2 ATTORNEYS Feb. 6, 1968 MASANAO MATSUI ETAL 3,367,945

THIOUREA DERIVATIVES OF MITOMYCIN v Filed Dec. 1, 1965 V 6 Sheets-Sheet 3 w v FIG. 5

lax 452120 5 ATTORNEYS 1968 MASANAO MATSUI ETAL 3,367,945

THIOUREA DERIVATIVES OF MITOMYCIN I Filed Dec. 1, 1965 6 Sheets-Sheet 4.

2 INVENTORS ,J. a/M BYfl/fi M fill M ATTORNEY Feb. 6,1968 MASANAO MATSUI ETAL I 3,367,945

THIOUREA DERIVATIVES OF MITOMYCIN Filed Dec. 1, 196.5 6 Sheets-Sheet 5 9 1 INVENTOR ATTORNEYS 6, 1968 MASANAO MATSUI ETAL 3,367,945

THIOUREA DERIVATIVES OF MITOMYCIN Filed Dec. 1, 1965 e Sheds-Sheet 6 INVENTOR ATTORNEY United States Patent 3,367,945 THIOUREA DERIVATIVES OF MITOMYCIN Masanao Matsui, Tokyo, Izuru Yamamoto, Yokohamashi, Yoshinori Soeda, Hiratsuka-shi, and Shigetoshi Wakaki, Tokyo, Japan, assig'nors to Kyowa Hakko Kogyo Co., Ltd., Tokyo, Japan, a corporation of Japan Filed Dec. 1, 1965, Ser. No. 510,800 Claims priority, application Japan, May 18, 1965, 40/ 28,772 18 Claims. (Cl. 260-3263) The present invention relates to novel derivatives of mitomycin C, and more specifically to therapeutically useful la-thiourea derivatives of mitomycin C.

In 1956, mitomycin A and mitomycin B-compounds having anti-tumor potency as well as antibacterial activitywere isolated by Hata et al. from a culture medium of Streptomyces caespitosus. Subsequently, Wakagi et al. isolated mitomycin C from a culture medium of the same microorganism. It is known that mitomycin C, which also has anti-tumor and antibacterial activities, is one of the most potent of the known anti-tumor substances. However, its clinical utilization is restricted because of its relatively high toxicity. Thus, as is pointed out on page 687 of The Merck Index, Seventh Edition (1960), published by Merck & Co., Inc., Rahway, N1, mitomycin C has been used primarily against for advanced malignancies.

The chemical structure of mitomycin C has recently been determined to correspond to the formula c1120 CONHz OCH:

The compound can also be designated 7-amino-9amethoxy-mitosane.

It is a desideratum in this art to embody new compounds which retain the advantages of mitomycin C, e.g. possess the antibacterial potencies thereof, but are free of the disadvantage thereof, i.e. are of such reduced toxicity relative to mitomycin as effectively to broaden the possibilities of use thereof.

The present invention realizes this desideratum by embodying novel la-thiourea derivatives of mitomycin C which are useful inter alia for the purposes for which the latter is used and in essentially the same way, but with elimination of the aspect of undue, and thereby prejudicial, toxicity.

The new compounds are prepared by reacting mitomycin C with an isocyanate (11), thereby to yield the objective la-thiourea derivative (III), according to the following reaction scheme:

0 II orno o oNrn mN R-NC s I ,0 CH3 113C- H N 0 NH Patented Feb. 6, 1968 "ice ll CHrO C ONH:

(III) wherein R is alkyl with 1 to 6 carbon atoms (e.g. methyl, ethyl, n-propyl, isopropyl, n-butyl, isobutyl, pentyl, hexyl), cycloalkyl with 5 or 6 carbon atoms (cyclopentyl, cyclohexyl), phenyl, mononuclear carbocyclic aryl (phenyl, chlorophenyl, lower alkylphenyl such as methylphenyl and dimethylphenyl, phenyl(lower) alkyl such as benzyl, dichlorobenzyl, phenylethyl, etc.), binuclear carbocyclic aryl (wnaphthyl, fi-naphthyl), etc.

The reaction of the abovedndicated reaction scheme is advantageously effected in a medium which is a solvent for mitomycin C, e.g. dioxane, N,N-dimethylformamide, acetone, tetrahydrofurane, furane, etc., the isothiocyanate (II) being added to the solution of the mitomycin C in such solvent and the reaction being allowed to proceed at ambient temperature (e.g. about 20 to about 30 C.). A good yield of the objective la-thiourea derivative (III) is obtained in each case.

The new derivatives (III) show differences in infra-red absorption spectrum, ultraviolet absorption spectrum, and visible light absorption spectrum in comparison with mitomycin C and also different elementary analyses, as Well as clearly different behaviors in column chromatography and thin layer chromatography, the results clearly evidencing the unitary character of the several substances.

On the accompanying sheets of drawing:

FIG. 1 shows the infra-red absorption spectrum of 1aisobutyl-thiocarbamoyl mitomycin C (see Example 1 infra);

FIG. 2 shows the infra-red absorption spectrum of la-methyl-thiocarbamoyl mitomycin C (see Example 1 infra);

FIG. 3 shows the infra-red absorption spectrum of la-ethyl-thiocarbamoyl mitomycin S (see Example 3 infra);

FIG. 4 shows the infra-red absorption spectrum of la-n-butyl-thiocarbamoyl mitomycin S (see Example 4 infra);

FIG. 5 shows the infra-red absorption spectrum of 1a-cyclohexyl-thiocarbamoyl mitomycin C (see Example 5 infra);

FIG. 6 shows the infra-red absorption spectrum of labenzyl-thiocarbamoyl mitomycin C (see Example 6 infra);

FIG. 7 shows the infrared absorption spectrum of laphenylethyl-thiocarbamoyl mitomycin C (see Example 12 infra);

FIG. 8 shows the infra-red absorption spectrum of 1ap-chlorobenzyl-thiocarbonyl mitomycin C (see Example 8 infra);

FIG. 9 shows the infra-red absorption spectrum of 1a-3,4-dichlorobenzyl-thiocarbamoyl mitomycin C (see Example 9 infra);

FIG. 10 shows the infra-red absorption spectrum of 1a-fl-naphthyl-thiocarbamoyl mitomycin C (see Example 10 infra); and

FIG. 11 shows the infra-red absorption spectrum of 1a-u-naphthyl-thiocarbamoyl mitomycin C (see Example 11 infra).

The following examples set forth, by way of illustration but not of limitation, presently preferred typical embodiments of the invention. In these examples, parts by weight bear the same relation to parts by volume as do grams to milliliters.

Example 1 880 parts by weight of mitomycin C are dissolved in 50 parts by volume of anhydrous dioxane in a receptacle of 100 parts by volume capacity. To the resultant solution, there is added a solution of 1520 parts by Weight of isobutyl-isothiocyanate in 8 parts by volume of anhydrous dioxane, and the mixture is allowed to stand at room temperature (about 25 C.) for 48 hours after first thoroughly agitating and uniformly intermixing the same at the said temperature.

The resultant reaction mass (solution) is then concentrated to substantial dryness on a water bath at 5557 C. under reduced pressure, the obtained residue is dissolved in anhydrous dioxane, using only just enough of the latter to effect dissolution, and anhydrous benzene or n-hexane is added to precipitate the objective la-isobutylthiocarbarnoyl mitomycin C, which is then isolated by filtration. The obtained substance is redissolved in anhydrous dioxane and again precipitated with anhydrous benzene or n-hexane, followed by filtration. The thusobtained 1a-isobutyl-thiocarbamoyl mitomycin C is washed with anhydrous benzene or n-hex'ane, and then dried.

Recovery, 86%.

Molecular formula C H O N S; molecular weight 449.3. Calculated values: C, 53.98%; H, 5.80%; N, 15.63%. Analyzed values: C, 53.32%; H, 6.08%; N, 12.75%. Ultraviolet and visible light absorption spectra:

m, 215 my (6: 33200), 358 m (62 19300), 500 m Example 2 Recovery, 86%.

Molecular formula C H O N S; molecular weight 407.3. Calculated values: C, 59.11%; H, 4.92%; N, 17.24%. Analyzed values: C, 53.31%; H, 5.22%; N, 13.59%. Ultraviolet and visible light absorption spectra:

x332 215 m (6: 24000), 256-7 m (6: 10000), 3589 mu (6: 16800), 55060 m (e: 145).

Example 3 The procedure according to Example 1 is repeated mutatis mutandis, the isobutyl-isothiocyanate being replaced by the equivalent amount of ethyl-isothiocyanate, whereby the obtained product is la-ethyl-thiocarbamoyl mitomycin C.

Recovery, 98%.

Molecular formula C H O N S; molecular weight 421.3. Calculated values: C, 51.43%; H, 5.23%; N, 16.67%. Analyzed values: C, 51.61%; H, 6.22%; N, 13.15%. Ultraviolet and visible light absorption spectra:

w 256-7 my. (6: 15800), 358-9 mu (e:

max.

Example 4 A2185 215 m (6: 31500), 2.367 my (6: 21200), 3589 Example 5 The procedure according to Example 1 is repeated mutatis mutandis, the isobutyl-isothiocyanate being replaced by the equivalent amount of cyclohexyl-isothiocyanate, whereby the obtained product is la-cyclohexylthiocarbamoyl mitomycin C.

Recovery, 88%.

Molecular formula C H O N S; molecular weight 475.3. Calculated values: C, 55.70%; H, 5.90%; N, 14.77%. Analyzed values: C, 55.20%; H, 6.34%; N, 12.90%. Ultraviolet and visible light absorption spectra:

x3532 215 my. (6: 32500), 2567 my (6: 22000), 358*9 m (e: 22800), 550-60 my (6: 232).

Example 6 The procedure according to Example 1 is repeated mutatis mutandis, the isobutyl-isothiocyanate being replaced by the equivalent amount of benzyl-isothiocyanate, whereby the obtained product is la-benzyl-thiocarbamoyl mitomycin C.

Recovery, 78.7%.

Molecular formula C H O N S; molecular weight 483.4. Calculated values: C, 57.27%; H, 4.98%; N, 14.52%. Analyzed values: C, 56.35%; H, 5.40%; N, 11.90%. Ultraviolet and visible light absorption spectra:

m, 215 my (5: 52100), 3589 m (61 22600), 550-60 Example 7 The procedure according to Example 1 is repeated mutatis mutandis, the isobutyl-isothiocyanate being replaced by the equivalent amount of phenyl-isothiocyanate, whereby the obtained product is la-phenyl-thiocarbamoyl mitomycin C.

Recovery, 98.0%.

Molecular formula C H O N S; molecular Calculated values: C, 57.51%; H, 4.58%; N, 14.58%.

Analyzed values: C, 57.67%; H, 5.28%; N, 12.01%.

Ultraviolet and visible light absorption spectra:

weight m, 215 m (e: 22700), 3589 m (e: 20200), 550-60 Example 8 The procedure according to Example 1 is repeated mutatis mutandis, the isobutyl-isothiocyanate being replaced by the equivalent amount of p-chlorophenyl-isothiocyanate, whereby the obtained product is la-p-chlorophenyl thiocarbamoyl mitomycin C.

Recovery, 38.0%.

Molecular formula C H O N SCl; molecular weight Calculated values: C, 52.40%; H, 4.36%; N, 11.90%;

Analyzed values: C, 51.03%; H, 4.37%; N, 12.42%; Cl,

Ultraviolet and visible light absorption spectra:

A 215 my (6: 22100), 256-7 my (6: 14250), 358-9 m max.

Example 9 The procedure according to Example 1 is repeated mutatis mutandis, the isobutyl-isothiocyanate being replaced by the equivalent amount of 3,4-dichlorobenzylisothiocyanate, whereby the obtained product is 1a3,4- dichlorobenzyl-thiocarbamoyl mitomycin C.

Recovery, 80.0%.

Molecular formula C H O N SCl molecular weight Calculated values: C, 49.93%; H, 4.34%; N, 12.66%;

Analyzed values: C, 49.11%; H, 3.89%; N, 12.95%.

Ultraviolet and visible light absorption spectra:

A 215 my. (6: 45200), 256-7 mp (62 18000), 358-9 mu max.

Example 10 The procedure according to Example 1 is repeated mutatis mutandis, the isobutyl-isothiocyanate being replaced by the equivalent amount of B-naphthyl-isothiocyanate, whereby the obtained product is 1a-B-naphthylthiocarbamoyl mitomycin C.

Recovery, 90.0%.

Molecular formula C H O N S; molecular weight 518.6. Calculated values: C, 60.25%; H, 4.63%; N, 13.51%. Analyzed values: C, 59.15%; H, 5.61%; N, 10.72%. Ultraviolet and visible light absorption spectra:

mm 215 my (6: 64800), 256-7 my (6: 13500), 358-9 m (e: 23200), 550-60 my. (6: 238).

Example 11 The procedure according to Example 1 is repeated mutatis mutandis, the isobutyl-isothiocyanate being replaced by the equivalent amount of a-naphthyl-isothiocyanate, whereby the obtained product is la-a-naphthylthiocarbamoyl mitomycin C.

Recovery, 90.0%.

Molecular formula C H O N S; molecular Weight 518.6. Calculated values: C, 60.25%; H, 4.63%; N, 13.51%. Analyzed values: C, 59.46%; H, 5.65%; N, 11.56%. Ultraviolet and visible light absorption spectra:

m, 215 111,1].(6171600), 258-9 my (6: 18200), 550-60 ma Example 12 The procedure according to Example 1 is repeated mutatis mutandis, the isobutyl-isothiocyanate being replaced by the equivalent amount of phenylethyl-isothiocyanate, whereby the obtained product is la-phenylethyl thiocarbamoyl mitomycin C.

Recovery, 97.0%.

Molecular formula C24H2'7O5N5S; molecular weight 497.4.

Calculated values: C, 58.07%; H, 5.44%; N, 14.11%.

Analyzed values: C, 58.71%; H, 5.34%; N, 12.85%; S,

Ultraviolet and visible light absorption spectra:

A 256-7 my. (e: 14500), 358-9 m (62 21600), 550-60 max.

Example 13 The procedure according to Example 1 is repeated mutatis mutandis, the isobutyl-isothiocyanate being replaced by the equivalent amount of 2,4-dimethylphenylisothiocyanate, whereby the obtained product is let-2,4- dimethyl-thiocarbamoyl mitomycin C.

Recovery, 80.0%.

Molecular formula C H O N S; molecular weight Calculated values: C, 59.06%; H, 5.11%; N, 13.78%.

Analyzed values: C, 60.87%; H, 5.99%; N, 10.98%.

Example 14 The procedure according to Example 1 is repeated mutatis mutandis, the isobutyl-isothiocyanate being replaced by the equivalent amount of 3-(o-methylphenyl)- propyl isothiocyanate, whereby the obtained product is 1a-3-(o -methylphenyl) propylthiocarbamoyl mitomycin C.

Recovery, 88.0%.

Molecular formula C H O N S; molecular weight Calculated values: C, 59.45%; H, 5.90%; N, 13.33%.

Analyzed values: C, 59.12%; H, 6.38%; N, 11.92%.

Ultraviolet and visible light absorption spectra:

AMeOH m, 215 my (6: 22100), 256-7 m (6: 14250), 358-9 my The toxicities of the 1a-thio-urea derivatives (III) of the present invention are reduced and are as follows in The minimum inhibiting concentrations of these derivatives against various kinds of bacilli examined by the agar plate culture method are as shown in Table 2:

TABLE 2 The minimum inhibiting concentration (mcg./ml.=micrograms per milliliter) of (meg/ml.)

0 ll OHgOCONHz (III) against various kinds of bacilli:

TABLE 2C0utinued la-isobutylla-methyl la-ethyl Name of Substance thiocarbamoyl thiocarbamoyl thiocarbamoyl mitomyein C mitomycin C mitomycin C Staphylococcus aureus 3.12 1. 56 6. 25 Sarcina lutea 12. 5 6. 12.5 Bacillus subtilis- 12 5 3. 12 6. 25 Salmonella typi 50 50 Shigella flezncri. 50 50 50 K'Zcbsiella pneumoniac- 50 50 50 Proteus vulgaris. 50 50 50 Escherichia colt"... 50 50 50 Bacillus pyocyaneous. 50 50 50 Vihrio comma.. 1. 56 0.78 1. 56 M ycvbactcrium tuberc'ul 12. 5 6 25 6. 25 Streptococcus haemolyticus. 12. 6 25 1. 56 Streptococcus faecolis 12.5 12.5 6. 25 Diplococcus pneamoniae 0.195 0. 195 0.097 Coryuebacterium diphtheriae 3.12 6. 25 1. 56

la-n-butyl ln-cyclohexyl labenzyl Name of Substance thiocarbamoyl thlocarbamoyl thiocarbamoyl mitomycin C mitomyein C mltomycln C Staphylococcus aureus 1. 56 3. 12 3. 12 Sarciua lute 3. 12 6. 25 6.25 Bacillus subtili 1. 56 3. 12 6. 25 Salmonella typi. 25 25 50 Shigella flezueri. 25 50 50 Klebsiella pntumon 50 50 50 Proteus vulgaris. 50 50 50 Escherichia coli 25 50 50 Bacillus pyocyartcus. 50 50 50 Vibrio com'ma 0. 39 0. 39 1. 56 M ycobacterium tuberculosis 3. 12 3. 12 12. 5 Streptococcus haemolyticus 1. 56 1. 56 1. 56 Streptococcus faecalis 3. 12 6. 25 6.25 Diplococcus pneumoniae 0.097 0. 195 0. 195 Corynebacterium diphtheriae 1. 56 0. 78 6. 25

la'phenylethyl la-3,4-d1- Name of Substance thiocarbamoyl chlorobenzyl mitomycin C thiocarbamoyl mitomycln G Staphlococcus aurcus 0.78 1. 56 Sarcina lutea. O. 39 1. 56 Bacillus subtilis. 0.78 3.12 Salmonella typi. 12.5 50 Shigella fleznerL 6. 25 50 Klebsiella pneumomae 25 50 Proteus vulgaris. 12.5 50 Escherichia coli 12.5 50 Bacillus pyocyaneus 12.5 50 Vibrio comma 0.097 12. 5 Mycobacterium tuberculosis 0. 39 6. 25 Streptococcus hacrnolyticus 0.39 1. 56 Streptococcus faecalis 1. 56 50 Diplococcus pneumoniae 0. 048 0.39 Coryuebactcrium diphtheriae 0. 39 1. 56

la-bnaphthyl law-naphthyl Name of Substance thioearbamoyl thioearbnmoyl mitomycin C mitomycin C Staphylococcus aureus 0.78 0.78 Sarcina luteo 0. 78 0. 78 Bacillus subtilis- 0.78 0. 78 Salmonella typi. 3.12 3. 12 Shioclla flez'heri 12.5 3.12 Klebsiella pneumoniae 12.5 12.5 Proteus vulgaris 12.5 12. 5 Escherichia coli 6. 25 6. 25 Bacillus pyocyaneusfl 50 25 Vtbrio comma 0. 097 0.097 M ycobacterium tuberculosis. 0. 78 0. 78 Streptococcus haemolyticus- 0.39 0. 79 Streptococcus faecalis 1. 56 1. 56 Diplococcus pneumoniae 0. 024 0. 048 Corynebacterium diphtheriae 0.39 0.195

Llke mltomycln C 1tself, the new la-tlnourea denva- Example 15 1 of P mvfmtilon are useful m vltYo One gram of 1a-lsobutyl-thlocarbamoyl 'mitomycm C is as antlseptlcs, me. for dlslnfectmg, and are also useful 1n homogeneously incorporated into about 500 grams of an combatmg toplcal mfectlons due to pathogenlc bactena, ointment base (Vase1ine) Repeated application of the e.g. 1n cases of staphylodermatltrs and the hke, and 1m thusmrepamd Ointment to topical infections due to th1s connection y y be PP toplcally 0r admlrlls- Staphylococcus auraus exhibits a curative effect. tered 1nternal1y (e.g. orally). The Vasehne may be replaced by any other suitable and Examples of formulatrons-solely lllustratlve and not dCSlI'Bd base, e.g. a vanishing cream base. The la-isobutylat all l1m1tat1ve-usmg the compounds (III) are a thlocarbamoyl m1ton1yc1n C may be replaced by any other follows: of the compounds III of th1s 1nvent1on with like elfect.

9 Example 16 0 H orno o o Nm wherein R is a member selected from the group consisting of alkyl with l to 6 carbon atoms, cycloalkyl containing 5 to 6 carbon atoms, phenyl, chlorine-substituted phenyl, lower alkylphenyl, phenyl(lower) alkyl, chlorinesubstituted phenyl(lower)alkyl and naphthyl.

2. A compound according to claim 1, namely, la-lower alkyl-thiocar bamoyl mitomycin C.

3. A compound according to claim 1, namely, 1aisobutyl-thiocarbamoyl mitomycin C.

4. A compound according to claim 1, namely, 1amethyl-thiocarbamoyl mitomycin C.

5. A compound according to claim 1, namely, 1aethyl-thiocarbamoyl mitomycin C.

6. A compound according to claim 1, namely, la-nbutyl-thiocarbamoyl mitomycin C.

7. A compound according to claim 1, namely, 1a cyclopentyl-thiocarbamoyl mitomycin C.

8. A compound according to claim 1, namely, cyclohexyl-thiocarbamoyl mitomycin C.

9. A compound according to claim 1, namely, phenyl(lower)alkyl-thiccarbamoyl mitomycin C.

10. A compound according to claim 1, namely, benzyl-thiocarrbamoyl mitomycin C.

11. A compound according to claim 1, namely, 1aphenylethyl-thiocarbamoyl mitomycin C.

12. A compound according to claim 1, namely, 1aphenyl-thiocarbamoyl mitomycin C.

13. A compound according to claim 1, namely, 1a-2,4- dimethylphenyl-thiocarbamoyl mitomycin C.

14. A compound according to claim 1, namely, 1a-3- (o-methylphenyl)-propyl-thiocarbamoyl mitomycin C.

15. A compound according to claim 1, namely, 121-pchlorophenyl-thiocar'barnoyl mitomycin C.

16. A compound according to claim 1, namely, 1a-,B- naphthyl-thiocarbamoyl mitomycin C.

17. A compound according to claim 1, namely la-unaphthyl-thiocar-bamoyl mitomycin C.

1-8. A compound according to claim 1, namely, 1a-3,4- dichlorobenzyl-thiocarbamoyl mitomycin C.

Burger: Medicinal Chemistry (2nd ed.) (1960), pp. 1134-35.

ALEX MAZEL, Primary Examiner.

I. A. NARCAVAGE, Assistant Examiner. 

1. COMPOUND OF THE FORMULA 